PROJECT SUMMARY/ ABSTRACT We have made the unexpected discovery that fermentation contributes to Salmonella's antioxidant defenses, an observation with wide ranging implications for defense against oxidative stress, well beyond bacteria. Infectious diarrhea afflicts a billion people a year and is responsible for 4% of all human deaths. Many of these infections are caused by one of the 2,500 serovars of nontyphoidal Salmonella enterica, which can inflict life- threatening systemic complications in the very young, very old, and HIV-infected individuals. Oxidative stress emanating from the enzymatic activity of the NADPH oxidase is one of the most potent host defenses Salmonella face during their associations with professional phagocytic cells. Genotoxicity that ensues from Fenton-mediated DNA double strand breaks together with cellular malfunctions associated with the oxidation of cysteine residues and metal cofactors in proteins constitute the paradigm for how oxidative stress kills Salmonella and numerous other bacterial pathogens. However, despite their central role in resistance to salmonellosis, the relative importance of the various mechanisms by which reactive oxygen species inflict anti- Salmonella activity is poorly understood. Our understanding of the adaptive responses that protect Salmonella against oxidative stress is similarly superficial. A screen of mutants in response to hydrogen peroxide, one of the most important effectors of the NADPH oxidase, revealed previously unanticipated roles for central metabolism and the electron transport chain in the hydrogen peroxide-mediated killing of Salmonella. Our preliminary data suggest oxidation of cell envelope proteins and plasmolysis-like lesions (i.e., separation of inner and outer membranes) as previously unsuspected steps in the killing of Salmonella during oxidative stress. These investigations offer an innovative framework for how NADPH oxidase inflicts potent anti-Salmonella activity during the innate response of macrophages. We will test the hypothesis that fermentation contributes to Salmonella's antioxidant defenses by assisting with ATP synthesis, balancing redox, and enabling disulfide bond formation in periplasmic proteins, thereby protecting the cell envelope from lethal damage by reactive oxygen species generated by the NADPH oxidase. Specifically, we will characterize the role fermentation plays in the antioxidant defenses of typhoidal and nontyphoidal Salmonella, elucidate the mechanism by which oxidative stress promotes fermentation, and determine how intracellular Salmonella is killed by the NADPH oxidase. Not only will this knowledge illuminate key aspects of Salmonella pathogenesis, but should also provide insights into unique and shared antioxidant defenses of various Salmonella serovars. Our research could ultimately have an impact on fields as diverse as microbial pathogenesis, aging, diabetes, or cancer biology for which oxidative stress is an intrinsic component. Drugs that specifically inhibit bacterial glycolytic enzymes and fermentative pathways may lead to the development of novel antibiotic treatments. Future Salmonella countermeasures could also explore strategies that increase respiratory activity as a means to foment oxidative killing.